scholarly journals Reverse Transcriptase-PCR Analysis of Bacterial rRNA for Detection and Characterization of Bacterial Species in Arthritis Synovial Tissue

2000 ◽  
Vol 68 (10) ◽  
pp. 6012-6026 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Michael Hurle ◽  
Anthony Wong ◽  
Scott Wilkie ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Bacterial Species ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Modified Technique ◽  
Culture Methods ◽  
Reverse Transcriptase Pcr

ABSTRACT Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.

scholarly journals Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

2000 ◽  
Vol 148 (6) ◽  
pp. 1107-1114 ◽  
Author(s):  
Roberto Giordano ◽  
Anna Rosa Magnano ◽  
Germana Zaccagnini ◽  
Carmine Pittoggi ◽  
Nicola Moscufo ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Pcr Amplification ◽  
Reverse Transcriptase Activity ◽  
Pcr Analysis ◽  
Immunogold Electron Microscopy ◽  
Direct Pcr ◽  
Vitro Fertilization ◽  
Rna Molecules ◽  
Exogenous Rna

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

Dominant symbiotic bacteria associated with wild medfly populations reveal a bacteriocin-like killing phenotype: a ‘cold-case’ study

2019 ◽  
Vol 110 (4) ◽  
pp. 457-462
Author(s):  
Silvia Ciolfi ◽  
Laura Marri
Keyword(s):  
Bacterial Species ◽  
Klebsiella Oxytoca ◽  
Pcr Amplification ◽  
Fruit Trees ◽  
Antagonistic Activity ◽  
Culture Collection ◽  
Symbiotic Bacteria ◽  
Bacterial Populations

AbstractThe gut of the agricultural pest Ceratitis capitata hosts a varied community of bacteria, mainly Enterobacteriaceae, that were implicated in several processes that increase the fitness of the insect. In this study, we investigated the antagonistic activity in vitro of Klebsiella oxytoca strains isolated in the 1990s from the alimentary tract of wild medflies collected from different varieties of fruit trees at diverse localities. Assays were carried out against reference strains (representative of Gram-negative and -positive bacterial species) of the American Type Culture Collection (ATCC). Eight Klebsiella, out of 11, expressed a killing activity against Escherichia coli ATCC 23739, and Enterobacter cloacae ATCC 13047; among the eight strains, at least one showed activity against Salmonella typhimurium ATCC 23853. Genomic DNA derived from all Klebsiella strains was then subjected to PCR amplification using specific primer pairs designed from each of the four bacteriocin (KlebB, C, D, CCL) sequences found so far in Klebsiella. KlebD primer pairs were the only to produce a single product for all strains expressing the killing phenotype in vitro. One of the amplicons was cloned and sequenced; the DNA sequence shows 93% identity with a plasmid-carried colicin-D gene of a strain of Klebsiella michiganensis, and 86% identity with the sequence encoding for the klebicin D activity protein in K. oxytoca. Our work provides the first evidence that dominant symbiotic bacteria associated with wild medfly populations express a killing phenotype that may mediate inter and intraspecies competition among bacterial populations in the insect gut in vivo.

scholarly journals Detection of Mycobacterium tuberculosis Group Organisms in Human and Mouse Joint Tissue by Reverse Transcriptase PCR: Prevalence in Diseased Synovial Tissue Suggests Lack of Specific Association with Rheumatoid Arthritis

2001 ◽  
Vol 69 (3) ◽  
pp. 1821-1831 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Andrew A. McColm ◽  
Julie A. Bagshaw ◽  
Richard Reece ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mycobacterium Tuberculosis ◽  
Synovial Fluid ◽  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Rt Pcr ◽  
Diverse Range ◽  
Susceptible Host ◽  
Joint Tissue ◽  
Reverse Transcriptase Pcr

ABSTRACT Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model ofM. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosisinfection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

Comparison of storage methods for reverse-transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples

2004 ◽  
Vol 116 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Christopher A Whittier ◽  
William Horne ◽  
Barrett Slenning ◽  
Michael Loomis ◽  
Michael K Stoskopf
Keyword(s):  
Reverse Transcriptase ◽  
Pcr Amplification ◽  
Gorilla Gorilla ◽  
Fecal Samples ◽  
Reverse Transcriptase Pcr ◽  
Storage Methods

Inhibition of proinflammatory genes in anti-GBM glomerulonephritis by targeted dexamethasone-loaded AbEsel liposomes

2008 ◽  
Vol 294 (3) ◽  
pp. F554-F561 ◽  
Author(s):  
Sigrídur A. Ásgeirsdóttir ◽  
Peter J. Zwiers ◽  
Henriëtte W. Morselt ◽  
Hendrik E. Moorlag ◽  
Hester I. Bakker ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Endothelial Cells ◽  
Targeted Delivery ◽  
Therapeutic Strategy ◽  
Disease Onset ◽  
Endothelial Activation ◽  
Pcr Analysis ◽  
Endothelial Cell Marker ◽  
Cell Adhesion Molecule 1

E-selectin-directed targeted drug delivery was analyzed in anti-glomerular basement membrane glomerulonephritis. Liposomes conjugated with anti-E-selectin antibodies (AbEsel liposomes) were internalized by activated endothelial cells in vitro through E-selectin-mediated endocytosis. At the onset of glomerulonephritis in mice, E-selectin was expressed on glomerular endothelial cells, which resulted in homing of AbEsel liposomes to glomeruli after intravenous administration. Accumulation of AbEsel liposomes in the kidney was 3.6 times higher than nontargeted IgG liposomes, whereas the accumulation of both liposomes in the clearance organs liver and spleen and in heart and lungs was comparable. In glomeruli, the AbEsel liposomes colocalized with the endothelial cell marker CD31. Quantitative RT-PCR analysis of laser-microdissected arterioles, glomeruli, and postcapillary venules demonstrated that targeted delivery of dexamethasone by AbEsel liposomes reduced glomerular endothelial expression of P-selectin, E-selectin, and vascular cell adhesion molecule-1 by 60–70%. The expression of these genes was not modulated in endothelial cells in nontargeted renal microvasculatures. Decrease of glomerular endothelial activation at disease onset was followed by reduced albuminuria at day 7. This study demonstrates the potential of vascular bed-specific drug delivery aimed at disease-induced epitopes on the microvascular endothelial cells as a therapeutic strategy for glomerulonephritis.

scholarly journals Development of a Microsphere-Based Serologic Multiplexed Fluorescent Immunoassay and a Reverse Transcriptase PCR Assay To Detect Murine Norovirus 1 Infection in Mice

2005 ◽  
Vol 12 (10) ◽  
pp. 1145-1151 ◽  
Author(s):  
Charlie C. Hsu ◽  
Christiane E. Wobus ◽  
Earl K. Steffen ◽  
Lela K. Riley ◽  
Robert S. Livingston
Keyword(s):  
Reverse Transcriptase ◽  
Laboratory Mouse ◽  
The United States ◽  
Mouse Serum ◽  
Murine Norovirus ◽  
Serum Samples ◽  
Diagnostic Assays ◽  
Reverse Transcriptase Pcr ◽  
Lethal Infection

ABSTRACT Murine norovirus 1 (MNV-1) is a newly recognized pathogen of mice that causes lethal infection in mice deficient in components of the innate immune response but not in wild-type 129 mice. In this study, in vitro-propagated MNV-1 was used as antigen to develop a multiplexed fluorescent immunoassay (MFI) to detect antibodies to MNV-1 in infected mice. The MNV-1 MFI was 100% specific and 100% sensitive in detecting anti-MNV-1 antibody in sera from experimentally infected mice. Testing of a large number of mouse serum samples (n = 12,639) submitted from contemporary laboratory mouse colonies in the United States and Canada revealed that 22.1% of these sera contained antibodies to MNV-1, indicating infection with MNV-1 is widespread in research mice. In addition, a reverse transcriptase PCR primer pair with a sensitivity of 25 virus copies was developed and used to demonstrate that MNV-1 RNA could be detected in the spleen, mesenteric lymph node, and jejunum from some experimentally infected mice 5 weeks postinoculation. These diagnostic assays provide the necessary tools to define the MNV-1 infection status of research mice and to aid in the establishment of laboratory mouse colonies free of MNV-1 infection.

Rotavirus G2P[4] Detection in Fresh Vegetables and Oysters in Mexico City

2014 ◽  
Vol 77 (11) ◽  
pp. 1953-1959 ◽  
Author(s):  
CAROLINA QUIROZ-SANTIAGO ◽  
CARLOS VÁZQUEZ-SALINAS ◽  
IVAN NATIVIDAD-BONIFACIO ◽  
BLANCA LILIA BARRÓN-ROMERO ◽  
ELSA IRMA QUIÑONES-RAMÍREZ
Keyword(s):  
Reverse Transcriptase ◽  
Viral Infections ◽  
Pcr Amplification ◽  
Detection Sensitivity ◽  
Gastrointestinal Infections ◽  
Reverse Transcriptase Pcr ◽  
Vegetable Samples ◽  
Fresh Vegetables ◽  
Contaminated Food ◽  
Combined Genotypes

Rotaviruses are the principal cause of dehydration caused by diarrhea in children younger than 2 years of age. Although these viral infections have mainly been associated with ingestion of fecally contaminated food and water, few studies have addressed the presence of the virus in food that is consumed raw or slightly cooked. In this work, 30 oyster samples and 33 vegetable samples were examined for the presence of rotavirus genotypes to evaluate their potential to produce gastrointestinal infections. The rotaviruses were identified by reverse transcriptase PCR amplification of the VP7 gene. G and P genotyping was also performed by reverse transcriptase PCR, with a detection sensitivity of up to 15 PFU/ml. Rotaviruses were found in 17 (26.9%) of 63 samples (10 oysters and 7 vegetables). The G2 genotype was found in 11 (64.7%) of 17 of the rotavirus strains, and 16 (94.1%) of 17 had the P[4] genotype. The combined genotypes found most frequently were G2P[4] (10 [58.82%] of 17), GNTP[4] (6 [35.29%] of 17), and G2P[NT] (1 [5.8%] of 17).

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